The Integration of FLIM and FRET for the Visualization of Flow-induced Molecular Hierarchies The research activity of the PI's laboratory is focused on the integration of cutting edge technologies for the elucidation of molecular mechanism in vascular biology, with the ultimate goal of enhancing our knowledge on cardiovascular functions and contributing to the health and well-being of humankind. In particular, novel imaging biosensors and technologies will be developed and employed to visualize the signaling transduction with high spatiotemporal resolutions in live cells under different mechanical forces, e.g. shear stress. In the long run, we aim to apply the knowledge and insights gained via these novel technologies for the development of new strategies and reagents to treat cardiovascular diseases, including thrombosis and atherosclerosis. Along this line, we proposed to apply biosensors based on fluorescence resonance energy transfer (FRET) to visualize the Src activity and its related molecular hierarchy at sub-cellular levels during mechanotransduction, which is funded by NHLBI (R01HL098472). Since FAK activity and its autophosphorylation at tyrosine 397 are critical for the Src activation, we plan to investigate the spatiotemporal coupling between FAK and Src activations in endothelial cells under mechanical stimulation. Recently, we have developed a new FRET pair, mOrange2 and mCherry, which contains colors distinct from the popular FRET pair, CFP and YFP. As such, two different molecular events, such as the activities of FAK and Src, can be simultaneously monitored in the same live cell with biosensors based on mOrange2/mCherry and CFP/YFP. However, there is a certain overlap between the excitation spectra of mOrange2 and mCherry to cause a non-specific cross- excitation of mCherry, which can cause artifacts and lower the sensitivity of FRET biosensors. Fluorescence lifetime imaging microscopy (FLIM) could help to solve this problem because the emission lifetime measurement of the donor mOrange2 alone is sufficient to deduce the FRET efficiency, without the need to measure the emission lifetime of the acceptor mCherry. The main objective of the current proposal is hence for the PI to obtain in-depth training in optics and fluorescence, particularly FLIM and its associated instruments and imaging analysis. Part of the training requires the PI to attend formal courses and training programs whereas the other aspects of training involve more self-studying and interactions with collaborators. Accordingly, three specific aims are proposed for the research plan: (1) develop a mOrange2/mCherry-based FAK biosensor and characterize it by FLIM; (2) visualize the spatiotemporal map of FAK activity under different flows with FLIM; (3) simultaneously monitor FAK and Src activities under different flows with FLIM. The information obtained will significantly advance our systematic understanding of the molecular mechanism by which different flows affect cardiovascular diseases, such as atherosclerosis and restenosis. The newly developed biosensors will also provide powerful tools for detecting cardiovascular diseases and assessing the efficacy of therapeutic inhibitors.